Relative Research On Molecular Diagnosis And Control Of Porcine Circovirus Type 2 Infection

Porcine circovirus type 2(PCV2) is one of the new animal viruses in recent years.It is associated with post-weaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC), congenital tremors(CT) and so on.PMWS is first emerged in western Canada in 1991 and is widespread in many countries by pathological and serological survey.Clinical signs include progressive weight loss,respiratory disorders,fever,pallor and jaundice,so PMWS has a serious economic impact on the swine industry worldwide.To find out the prevalence and pathogenicity of PCV2 infection in our country and effective control of porcine circovirus type 2 infection,molecular diagnosis,genetically engineering vaccine and effects of different selenium sources on the PCV2 replication in vitro were studied.So the following researches were explored:1.Multiplex Ploymerase Chain Reaction for Rapid Detection of Porcine Circovirus Type 2,Porcine Parvovirus and Pseudorabies VirusPorcine circovirus type 2(PCV2),Porcine parvovirus infection(PPV) and Pseudorabies virus(PRV) are important pathogenic factors to cause sows reproductive barriers.To investigate co-infection of these diseases from clinical samples and establish rapid differential diagnosis,a multiplex PCR(mPCR) assay was developed to simultaneously detect multiple viral infection of swine.Specific primers for each of three common DNA viruses,namely,PCV2,PPV and PRV were used for testing procedure and four specific bands of 269 bp(PCV2),583 bp(PPV) 372 bp(PRV gB)and147 bp(PRV gE)were amplified. The assay was shown to be highly sensitive when a composite of all three viruses were amplified including both field and gene-deleted permutations of PRV.No specific band was amplified from other pathogenic viruses and bacterium.As little as PCV2 10 ng,PPV 10^-6.2 TCID₅₀ PRVgB 10^-3.8TCID₅₀ and PRVgE10^-5.8TCID₅₀ were detected using agarose gel electrophoresis in this multiplex PCR.2.Development of Real-time Qantitative PCR for Detecting PCV2 with SYBR Green I A 107 bp region of the PCV2 ORF2 gene was cloned to pMD19-T vector,and the constructed plasmid was named pT-PCV2(107).Serial dilutions of plasmid pT-PCV2(107) were used to quantify the virus genomic copy number,and served as standards curve.The regression coefficient of the quantitative curve was 0.997 and the Tm of melting curve analysis is 81.53±0.16℃.By comparison of viral load in sample from 32 healthy animals and 32 PMWS animals,healthy pigs had viral load less than 10⁴ copies perμL and 44% (14/32) were tesed positive for PCV2,while all clinically sick PMWS pigs had PCV2 loads above 10⁵ copies perμL and 96.8%(31/32) were tested positive for PCV2.The developed real time quantitative PCR assay is a rapid and simple to monitor and detect PCV2.3.Development of Inderect ELISA Assay by Recombinant Protein Coded by ORF2 Gene for Detecting PCV2Accordingto the published whole genome sequence of PCV2,a pair of primers was designed to for cloning of ORF2 gene in PCV2 by PCR.The PCR product was linked with pMD18-T vector,cloned into expression vector pET-32a and transformed into E.coli Rosetta for the prokaryotic expression of the ORF2 gene.The expressed protein was purified by His-Bind purification Kit.Protein average concentration was 1.10 mg·mL^-1 and 1.8μg·mL^-1 purified protein was used as antigen to establish an ELISA assay for the detection of antibody in the serum of swine infected with PCV2.The ELISA assay is corresponded with diagnostic kit supplied by Beijing Yuanheng Company and the coincidence rate was 85%.The detection of serum samples of 36 sows and 156 10-160 day-old piglets from Changzhou by the ELISA revealed thaut the positive rate of sow serum reached 81.4%,while that of piglet serum first declined with the aging of the piglets and then climbed up due to the infection of PCV2.The indirect ELISA assay developed in the study could be used in massive detection of antibody to PCV2 due to its specificity, good repeatability and sensitivity.4.Construction and Identifieation of Recombinant Porcine Parvovirus-like Particles Formed by the Hybrid VP2 Protein Carrying Immunoreactive Epitope of PCV2PPV VP2 virus-like particles(VLPs) are non-replieative vectors for delivery of heterologous epitopes and induction of immune responses.A recombinant PPV virus-like particles(VLPs) using as antigen delivery system was formed by the hybrid VP2 protein carrying immunoreactive epitopes,residues 165 to 200 from the PCV2 capsid protein and expressed in adenovirus vector system.After transfection,recombined virus(rAd-â–³Cap-â–³ VP2) was developed.The specific 64 kDa band was showed by analysis of Western-blotting and specific fluorescence was observed by IFA which indicated that chimeric protein had expressed in HEK-293A cells.Moreover,the recombinant VP2 protein was to be similar to PPV in haemagglutinating activity.It was noteworthy that many self-assembled virus-like particles[PPV:VLP(PCV2)]were found in VP2 crude by electronmicroscope.5.Construction and Identification of Recombinant Adenoviruses Coexpression of IL-2 and Cap Protein of PCV2The cap protein contains virus main epitope and is PCV2 vaccine candidates.IL-2,an important adjuvant,can enhance vaccine effect.In the study,the cap gene and IL-2-Cap gene were amplified by PCR,and then cloned into the transfer vector pShuttle-CMV respectively.The recombinant plasmids and bone vector pAdEasy^TM were cotransformed into BJ5183 to generate the recombinant plasmids pAd-Cap and pAd-Cap-IL-2.The recombinant adenovirus(rAd-Cap and rAd-Cap-IL-2) were generated by transfection and plaque purification in HEK-293A cells.The transcription and expression of Cap protein andCap-IL-2 in infected 293 cells were confirmed by RT-PCR,IFA,indirect-ELISA,Western-blotting and IL-2 ELISA kit respectively.The study laid the foundation for development of the recombinant vaccine against PCV2.6.Immunogenicity of Recombinant Adenovirus in MiceIn this study,we have examined immunity of recombinant adenovirus rAd-â–³Cap-â–³VP2 expressing PPV:VLP-(PCV2),rAd-Cap and rAd-Cap-IL-2 in mice.One hundred and twenty ICR mice(6-week-old) were randomly assigned to four groups.Mice in group 1(n=30),group 2(n=30) and group 3(n=30) were inoculated subcutaneously with 10¹⁰ TCID₅₀/mouse of rAd-â–³Cap-â–³VP2,rAd-Cap and rAd-Cap- IL-2 respectively and boosted with same dose 2 weeks later.Group 4 mice(n=30) were inoculated with wild adenovirus and served as controls.At days 0,14,28,35,42,49 and 56 after the first immunization,four mice of each group were euthanized and the serum samples were collected from mice for detection of antibody to PCV2 by indirect enzyme -linked immunosorbent assay and virus neutralization assay.The result showed that mice in group 1,2 and 3 but group 4 could generate specific antibody to PCV2 with the levels gradually increasing to maximum titers of 1:10000,1:6000 and 1:9600 respectively detected by indirect ELISA and PCV2 neutralization antibody with neutralization titers up to 1:16,1:10 and 1:12 at 49 days after the first immunization.Meanwhile,PPV neutralization antibody titer can reach 1:20 in sera of mice inoculated with rAd-â–³Cap-â–³VP2.LTT showed that mice inoculated with the recombinant adeuovirus resulted in high LPS-/ConA-stimulation indices at 42 days after the first immunization.It can be concluded that rAd-â–³Cap-â–³VP2, rAd-Cap and rAd-Cap-IL-2 may induced humoral and cellular immune responses against PCV2,and immune effect of rAd-â–³Cap-â–³VP2 is the most obvious.7.Effects of Different Selenium Sources on the PCV2 Replication in VitroIn this study,the effects of sodium selenite,DL-Selenomethionine and Kappa-selenocarrageenan on PCV2 replication in vitro were studied by the established real-time quantitative PCR for detecting PCV2.The results indicated that the three selenium sources had different inhibitory effects on the PCV2 replication in the PK-15 cells. Among them,DL-Selenomethionine had obvious inhibitory effect.The higher the concentration is,the stronger inhibitory effects are when concentration of DL-Selenomethionine is between 2-16μmol·L^-1.The inhibition was concentrationdependent. In addition,the three selenium sources may enhance activation of GPX differently.Among them,DL-Selenomethionine is remarkable.The results showed that inhibitory effects of selenium on virus replication were accomplished mostly by eliminating free radical and resisting oxidation.